大鼠肝脏葡醛酸转移酶活性测定的显色光度法研究

Colorimetric Determination of UDP-Glucuronosyl Transferases Activity in Rat Liver

在探讨显色反应机理的基础上,对以2-氨基酚为底物测定葡醛酸转移酶(UGTs)活性的显色光度法进行了优化.结果表明,在控制反应条件下,过量的底物本身并不发生显色反应,而其葡醛酸结合代谢物2-氨基酚葡醛酸苷可发生重氮-偶氮化反应,在540nm波长处有最大吸收,且吸光度与代谢物浓度成正比.因此,可以具有相同发色团的苯胺为标准,根据标准曲线计算UGTs的酶活性.2-氨基酚葡醛酸苷在0.5～8μmol/L范围内线性良好,r>0.999(n=6),吸光度值可在显色完全后40 min内保持稳定.应用本法测定了雄性SD大鼠肝微粒体及S9组分的UGTs活性,结果分别为0.704 nmol·min-1·mg-1和0.177 nmol·min-1·mg-1.该方法测定条件的可控性好,操作简便,结果准确可靠,适用于临床前动物药代实验研究中UGTs活性的常规评价.

On the basis of investigation of coloring mechanism,, the colorimetric method for determination of UDP- glucuronosyl transferases （UGTs） activity with o-aminophenol as a substrate of enzymatic reaction has been optimized. Under the controlled conditions, not the remainder substrate but its glucuronidated metabolite could react with freshly prepared sodium nitrite and N-naphthylethylene, and then azo product is obtained with a maximum ab- sorption at 540 nm. Since this absorbance is in proportion to the concentration of glucuronidated metabolite, the activity of UGTs could be quantified by the calibration curve with aniline as a standard, which has the same chromophore as the glucuronidated metabolite of o-aminophenol. The calibration curve is linear for glucuronidated o-ami- nophenol in the range of 0. 5 - 8 μmol/L （ r 〉 0. 999 ）. The azo product is steady within 40 min after completion of the coloring reaction. With the present method the UGTs activities for male SD rat liver microsome and S9 are determined as 0. 704 nmol · min^-1 · mg^-1 protein and 0. 177 nmol · min^-1 · mg^-1 protein, respectively. It has been demonstrated that the present method is controllable, accurate, precise, and suitable for the routine determination of UGTs activity in preclinic pharmcokinetics study.